The ability to deliver nucleic acids, proteins, peptides, amino acids, small molecules, viruses, etc. (hereafter referred to collectively as “non-translocating moieties”) into cells or into specific cell types is useful for various applications in oncology, developmental biology, gene therapy and in the general understanding of the mode of operation of particular proteins, nucleic acids and small molecules in a model system. Most therapeutically important proteins and peptides do not readily translocate across biological membranes. However, some transactivating factors and homeoproteins have been shown to be capable of facilitating membrane translocation, including Tat derived peptides (Fawell et al., 1994 Proc. Natl. Acad. Sci. USA 91:664-668), the third helix of the antennapedia homeodomain protein (Derossi et al., 1994, J. Biol. Chem. 269:10444-10450; U.S. Pat. Nos. 5,888,762 and 6,015,787), and VP22 (Schwarze et al., 2000, Trends Pharmacol. Sci. 21:45-48). Such naturally derived peptides are often isolated in membrane vesicles within the cytoplasm of the cell, which often prevents the associated non-translocating moiety from accessing its desired target (Potocky et al. 2003, J Biol. Chem. 278: 50188-94).
To date, novel peptides have been engineered through the use of two different approaches. The first approach produces candidate peptides by chemically synthesizing a randomized library of 6-10 amino acid peptides (J. Eichler et al., 1995, Med. Res. Rev. 15:481-496; K. Lam, 1996, Anticancer Drug Des. 12:145-167; M. Lebl et al., 1997, Methods Enzymol. 289:336-392). In the second approach, candidate peptides are synthesized by cloning a randomized oligonucleotide library into an Ff filamentous phage gene, which allows peptides that are much larger in size to be expressed on the surface of the bacteriophage (H. Lowman, 1997, Ann. Rev. Biophys. Biomol. Struct. 26:401-424; G. Smith et al., 1993, Meth. Enz. 217:228-257). Randomized peptide libraries up to 38 amino acids in length have also been made, and longer peptides are likely achievable using this system. The peptide libraries that are produced using either of these strategies are then typically mixed with a pre-selected matrix-bound protein target. Peptides that bind are eluted, and their sequences are determined. From this information new peptides are synthesized and their biological properties are determined. Phage display has previously been used to identify translocating peptides, but relatively few peptides have been isolated by this method, and those that have are generally cell type specific and require endocytosis for entry into a cell (Gao et al. 2002, Bioorg Med. Chem., 10: 4057-65). One disadvantage associated with prior art peptides that rely on endocytosis to cross the cellular membrane is that typically such a mechanism results in the delivery of the translocating peptide, and any associated non-translocating moiety, to endosomes where they are both destroyed without causing the desired cellular effect.
A further disadvantage of the prior art is that the size of the libraries that can be generated with both phage display and chemical synthesis is limited to within the 106-109 range. This limitation has resulted in the isolation of peptides of relatively low affinity, unless a time-consuming maturation process is subsequently used. This library-size limitation has led to the development of techniques for the in vitro generation of peptide libraries including mRNA display (Roberts, & Szostak, 1997, Proc. Natl. Acad. Sci. USA, 94, 12297-12302), ribosome display (Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 9022-9026) and CIS display (Odegrip et al., 2004, Proc. Natl. Acad. Sci. USA, 101 2806-2810) amongst others. These libraries are superior to phage display libraries in that the size of libraries generated by such methods is 2-3 orders of magnitudes larger than is possible with phage display. This is because unlike techniques such as phage display, there are no intermediate in vivo steps.
However, at present no methods have been described using known in vitro display systems that allow for the specific and selective identification of membrane-translocating peptides (MTPs). Moreover, such methods could allow for the identification of MTPs that are capable of crossing layers of cells, such as endothelium.
Hence, there remains a need for methods that could provide a much needed advance in the field of MTP discovery and peptide drug development.